Project Summary/Abstract Enterococci such as Enterococcus faecalis are among the trillions of intestinal microbes that normally coexist in a symbiotic relationship with their host. However, antibiotic-resistant enterococci are also among the three most common causes of hospital-acquired infections and therefore represent a serious public health problem. An important risk factor for the acquisition of hospital-acquired enterococcal infections is prior therapy with antibiotics, such as broad-spectrum cephalosporins, to which enterococci are intrinsically resistant. Thus, the inherent abilities of enterococci to colonize the gut and withstand assault by antimicrobial agents are especially critical for pathogenesis of enterococcal infections. Yet, the mechanisms of enterococcal gut colonization and antimicrobial resistance are not fully understood. One critical determinant of these intrinsic traits is a transmembrane Ser/Thr kinase known as IreK, which belongs to a family of bacterial kinases containing extracellular PASTA domains. Such ?PASTA kinases? control critical processes including antibiotic resistance, toxin production, virulence, or cell division in nearly all Gram-positive bacteria; in some bacteria, a PASTA kinase is essential for viability. As such, PASTA kinases represent attractive targets for new therapeutics. However, a basic understanding of the structure and dynamics of PASTA kinases in solution, and the structure-function relationships that promote environmental sensing and coordination of biological responses in vivo, is lacking. Such information is critical to inform development of new therapeutic approaches. The research proposed here seeks to address this gap by elucidating fundamental structure-function relationships in solution for a representative PASTA kinase, the IreK kinase from E. faecalis, which we have shown is required for intrinsic resistance of E. faecalis to cell-wall-active antibiotics and to detergents present in bile. This collaborative research is designed to apply new approaches and perspectives to elucidate novel insights into the function of the IreK kinase using a complementary combination of in vivo functional assays and EPR spectroscopy approaches to determine the PASTA module conformation in solution and understand how kinase domain dynamics contribute to kinase activation. By successfully completing this work, we will obtain important new insights into a representative bacterial PASTA kinase to help define the structure-function relationships for an entire family of critical bacterial signaling proteins with the long-term goal of modulating the activity of these kinases therapeutically.